Application Information: SMAD2 (phospho Thr8) antibody

WB: Use at a concentration of 0.1 - 0.75 µg/ml. Detects a band of approximately 58 kDa. Optimal dilutions/concentrations should be determined by the end user.

Positive Controls

HepG2 cells treated with TGF

Concentration

0.35
mg/ml
(Please refer to the vial label for the specific concentration)

Purification

Immunogen affinity purified

Purification Note

The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated SMAD 2. The final product is generated by affinity chromatography using a SMAD

Specifications: SMAD2 (phospho Thr8) antibody

Full Name

SMAD family member 2

Product Description

Rabbit polyclonal to Smad2 ( phospho T8 )

Background

SMAD 2 is a 58 kDa member of a family of proteins involved in cell proliferation, differentiation and development. The SMAD family is divided into three subclasses: receptor-regulated SMAD's, activin/TGFa receptor-regulated (SMAD 2 and 3) or BMP receptor regulated (SMAD 1, 5, and 8); the common partner, (SMAD 4) that functions via its interaction to the various SMAD's; and the inhibitory SMAD's, (SMAD 6 and SMAD 7). SMAD 2 consists of two highly conserved domains, the N terminal Mad homology (MH1) and the C-terminal Mad homology 2 (MH2) domains. The MH1 domain binds DNA and regulates nuclear import and transcription while the MH2 domain conserved among all the SMAD's regulates SMAD 2 oligomerization and binding to cytoplasmic adaptors and transcription factors. Activated SMAD 2 associates with SMAD 4 and translocates as a complex into the nucleus, allowing its binding to DNA and transcription factors. This translocation of SMAD 2 (as well as SMAD 3) into the nucleus is a central event in TGF beta signaling. Phosphorylation of threonine 8 in the calmodulin binding region of the MH1 domain by extracellular signal regulated kinase 1(ERK 1) enhances SMAD 2 transcriptional activity, which is negatively regulated by calmodulin. The regulation of SMAD 2 phosphorylation on threonine 8 by ERK 1 and calmodulin is critical for SMAD 2 mediated signaling.

Lysates prepared from HepG2 cells stimulated with TGFbeta were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.35 µg/ml Smad2 [pT8] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG HRP-conjugate and bands were detected. The data show that the peptide corresponding to Smad2 [pT8] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Lysates prepared from HepG2 cells stimulated with TGFbeta were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.35 µg/ml Smad2 [pT8] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG HRP-conjugate and bands were detected. The data show that the peptide corresponding to Smad2 [pT8] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.